We continue to investigate the composition and molecular organization of the mechanotransduction (MET) channel complex in hair cell stereocilia. TMC1, TMC2, and TMIE are membrane proteins that are essential for MET and proposed to be integral components of the MET channel complex. Other adaptor or regulatory proteins have also been suggested to be part of the MET channel complex and capable of modulating its channel properties While it has been reported that MET channel properties in inner hair cells (IHCs) show little or no variation along the organ of Corti, measurements in outer hair cells (OHCs) performed in the laboratory of Robert Fettiplace (University of Wisconsin) show three-fold higher putative single channel conductances in OHCs at the base of the organ of Corti than at the apex. This tonotopic gradient in OHC single channel conductance is maintained in the absence of TMC2, but not in absence of TMC1, suggesting that TMC1, independent of TMC2, is capable of generating different channel conductances. This led us to hypothesize that variations in channel properties in individual MET channel complexes may result from a variation in the number of TMC1 proteins. To evaluate this, we quantified the expression of TMC1 at stereocilia tips along the organ of Corti using a previously reported transgenic mouse expressing Tmc1-mCherry. We found that the fluorescent intensity of TMC1-mCherry puncta at the stereocilia tips showed a linear, 3-fold increase from apex to base in OHCs. Conversely, only a small increase in TMC1-mCherry fluorescence in IHC stereocilia was observed. These data are consistent with the channel conductance measurements, supporting the idea that TMC1 levels are associated with generating the tonotopic gradient of MET channel complex properties. It is not yet known whether TMC proteins make up the pore component of the channel, nor is it clear how many TMC molecules are associated with each unitary channel. We are currently attempting to determine the number of TMC proteins per MET site. Our approach uses the bleaching and blinking properties of the fluorophore to calculate the number of molecules within each TMC fluorescence punctum at stereocilia tips. Our initial estimates suggest a range of 4-16 TMC molecules per MET site. This unexpectedly broad range of TMC molecules at each MET site raises new possibilities regarding the organization of the channel complex: 1) there is an excess of TMC molecules within each channel complex; or 2) each channel complex requires multiple TMCs; or 3) there are in fact multiple channels per MET site possibly operating in a coordinated manner. It has been shown that, functionally, TMC2 is able to replace TMC1, at least early postnatally. The channel properties when only TMC2 is present are different than when only TMC1 is present. To understand the underlying molecular basis, we sought to ascertain the levels of each TMC isoform in the absence of the other. We used Tmc1/ ;Tmc2/ mice with a mosaic expression of Tmc1-mCherry and Tmc2-AcGFP, so that hair cells expressed either TMC1-mCherry or TMC2-AcGFP, or neither or both, providing several side-by- side expression conditions, and internal controls. We used mice aged P6 when both isoforms are normally well expressed. Interestingly, we found that when hair cells did not express any TMC1-mCherry, the expression levels of TMC2-AcGFP within the stereocilia bundle was consistently higher than when both isoforms were expressed. The lack of TMC2-AcGFP from hair cells also led to an increase, albeit much smaller, in TMC1-mCherry levels. These data suggest that: 1) In the absence of TMC1, TMC2 is either upregulated or retained for a longer time at the MET site; and 2) the decreasing levels of TMC2 from stereocilia which normally begins at P7 may be promoted by the increasing levels of TMC1. Finally, we found that stereocilia bundle morphology was also influenced by lack of either or both TMC isoforms, highlighting a link between TMC expression, MET activity, and stereocilia bundle development and regulation. Specifically, stereocilia bundles from hair cells lacking both TMC1 and TMC2 showed a phenotype consistent with delayed development, including multiple rows of stereocilia and longer kinocilium. Expression of either TMC1 or TMC2 at this stage was sufficient for normal bundle morphology, although the overexpression of TMC2 (in the absence of TMC1), appeared to accelerate bundle development. This suggests that while both TMC1 and TMC2 expression is required for normal stereocilia bundle morphology, the two isoforms likely make differential contributions towards MET channel properties as well as the complex mechanisms that regulate the slope of the stereocilia staircase and overall bundle development. In summary, we propose isoform dependent roles for TMC1 and TMC2 in: 1) varying channel conductance across the tonotopic gradient and 2) regulating stereocilia bundle development. Our preliminary data on the number of TMC molecules per MET site will also shed new light on the molecular make-up of the channel complex. We are also examining the localization and quantifying the copy number of TMIE molecules at the MET complex sites along the organ of Corti functional tonotopic gradient, using a transgenic mouse line expressing TMIE-EGFP that we generated using the CRISPR/Cas9 approach. Preliminary results confirm expression of TMIE-EGFP at the MET sites in both organ of Corti and vestibular hair cells. TMIE-EGFP is also found in abundant levels along the length of stereocilia, in particular during bundle development. We are currently crossing mice expressing TMIE-EGFP with mice expressing TMC1-mCherry to examine the colocalization and coordinated traffic and turnover of these two key MET proteins. The molecular nature of the MET channel has long been elusive and it is still not clear whether TMIE or TMCs form the MET channel pore. Although normal MET current is lost in the absence of TMC1 and 2 or TMIE, it is replaced by an atypical mechanically sensitive current evoked by reversed-polarity displacement of the hair bundle. Furthermore, proteomics and genomics data suggests the possibility that other TMC isoforms (TMC4 and TMC5) may also be present in the organ of Corti and be part of the MET complex. We thus generated knockin mice expressing fluorescently-tagged TMC4 and TMC5 to determine spatiotemporal expression of these proteins in the organ of Corti. These studies are currently underway. We examined other tissues from the TMC4-GFP and TMC5-mCherry-expressing mice, and found that both proteins localize at the tips of microvilli of various epithelial cells. This result is particularly intriguing given the structural and molecular similarities between microvilli and stereocilia. Aside from both comprising a parallel actin bundle core, stereocilia and microvilli share the same, or homologous, actin bundling proteins (e.g., Espin and EPS8), membrane-cytoskeletal tether proteins (e.g., MYO1A), and extracellular proteins that connect adjacent stereocilia or microvilli (eg. protocadherins). Furthermore, in light of recent evidence suggesting that some microvilli could also mechanoresponsive, the presence of TMC4 and TMC5 at microvilli tips has important implications for these proteins in stereocilia. We plan to continue to use microvilli as an accessible and robust model system to understand the role of TMC4 and TMC5 and their possible functional similarities to TMC1 and TMC2.